Technical Field
This invention is generally directed to controlling the translocation of a target molecule for sensing by a nanopore, as well as methods and products relating to the same.
Description of the Related Art
Measurement of biomolecules is a foundation of modern medicine and is broadly used in medical research, and more specifically in diagnostics and therapy, as well in drug development. Nucleic acids encode the necessary information for living things to function and reproduce, and are essentially a blueprint for life. Determining such blueprints is useful in pure research as well as in applied sciences. In medicine, sequencing can be used for diagnosis and to develop treatments for a variety of pathologies, including cancer, heart disease, autoimmune disorders, multiple sclerosis, and obesity. In industry, sequencing can be used to design improved enzymatic processes or synthetic organisms. In biology, this tool can be used to study the health of ecosystems, for example, and thus has a broad range of utility. Similarly, measurement of proteins and other biomolecules has provided markers and understanding of disease and pathogenic propagation.
An individual's unique DNA sequence provides valuable information concerning their susceptibility to certain diseases. It also provides patients with the opportunity to screen for early detection and/or to receive preventative treatment. Furthermore, given a patient's individual blueprint, clinicians will be able to administer personalized therapy to maximize drug efficacy and/or to minimize the risk of an adverse drug response. Similarly, determining the blueprint of pathogenic organisms can lead to new treatments for infectious diseases and more robust pathogen surveillance. Low cost, whole genome DNA sequencing will provide the foundation for modern medicine. To achieve this goal, sequencing technologies must continue to advance with respect to throughput, accuracy, and read length.
Over the last decade, a multitude of next generation DNA sequencing technologies have become commercially available and have dramatically reduced the cost of sequencing whole genomes. These include sequencing by synthesis (“SBS”) platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and analogous ligation based platforms (Complete Genomics, Life Technologies Corporation). A number of other technologies are being developed that utilize a wide variety of sample processing and detection methods. For example, GnuBio, Inc. (Cambridge, Mass.) uses picoliter reaction vessels to control millions of discreet probe sequencing reactions, whereas Halcyon Molecular (Redwood City, Calif.) was attempting to develop technology for direct DNA measurement using a transmission electron microscope.
Nanopore based nucleic acid sequencing is a compelling approach that has been widely studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides as they were electrically translocated through an alpha hemolysin nanopore embedded in a lipid bilayer. It was demonstrated that during polynucleotide translocation partial blockage of the nanopore aperture could be measured as a decrease in ionic current. Polynucleotide sequencing in nanopores, however, is burdened by having to resolve tightly spaced bases (0.34 nm) with small signal differences immersed in significant background noise. The measurement challenge of single base resolution in a nanopore is made more demanding due to the rapid translocation rates observed for polynucleotides, which are typically on the order of 1 base per microsecond. Translocation speed can be reduced by adjusting run parameters such as voltage, salt composition, pH, temperature, and viscosity, to name a few. However, such adjustments have been unable to reduce translocation speed to a level that allows for single base resolution.
Stratos Genomics has developed a method called Sequencing by Expansion (“SBX”) that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer called an “Xpandomer” (Kokoris et al., U.S. Pat. No. 7,939,259, “High Throughput Nucleic Acid Sequencing by Expansion”). The transcribed sequence is encoded along the Xpandomer backbone in high signal-to-noise reporters that are separated by ˜10 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. Xpandomers can enable several next generation DNA sequencing detection technologies and are well suited to nanopore sequencing.
Gundlach et al. (Proc. Natl. Acad. Sci. 107(37): 16060-16065, 2010) have demonstrated a method of sequencing DNA that uses a low noise nanopore derived from Mycobacterium smegmatis (“MspA”) in conjunction with a process called duplex interrupted sequencing. In short, a double strand duplex is used to temporarily hold the single stranded portion in the MspA constriction. This process enables better statistical sampling of the bases held in the limiting aperture. Under such conditions single base identification was demonstrated; however, this approach requires DNA conversion methods such as those disclosed by Kokoris et al. (supra).
Akeson et al. (WO2006/028508) disclosed methods for characterizing polynucleotides in a nanopore that utilize an adjacently positioned molecular motor to control the translocation rate of the polynucleotide through or adjacent to the nanopore aperture. At this controlled translocation rate (350-2000 Hz (implied measurement rate)), the signal corresponding to the movement of the target polynucleotide with respect to the nanopore aperture can be more closely correlated to the identity of the bases within and proximal to the aperture constriction. Even with molecular motor control of polynucleotide translocation rate through a nanopore, single base measurement resolution is still limited to the dimension and composition of the aperture constriction. As such, in separate work, Bayley et al. (alpha hemolysin: Chemistry & Biology 9(7):829-838, 2002) and Gundlach et al. (MspA: Proceedings of the National Academy of Sciences 105(52):20647-20652, 2008) have disclosed methods for engineering nanopores with enhanced noise and base resolution characteristics. However, a demonstration of processive individual nucleotide sequencing has yet to be published that uses either (or both) a molecular motor for translocation control and an engineered nanopore. Current state of the art suggests that signal deconvolution of at least triplet base sets would be required in order to assign single base identity.
Nanopores have proven to be powerful amplifiers, much like their highly exploited predecessors, Coulter Counters. However, the current generation of organic nanopores (such as Hemolysin and MspA), that have been tasked with base recognition of DNA, are transmembrane proteins that do not interact with DNA in nature. They do not have natural functions for controlling DNA translocation. As has been discussed, this is a recognized shortcoming that some have attempted to correct by adding functionality with protein motors adjacent to the nanopores. For example, Akeson's group added phi 29 polymerase adjacent to the alpha-hemolysin nanopore so that ss-DNA could be fed into the pore at a controlled rate (see G. M. Cherf et al. “Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision,” Nat Biotech, vol. advance online publication, February 2012). This approach complicates the assay and imposes a separation of the measurement region in the alpha hemolysin from the position control in the polymerase that can introduce additional noise and sequence dependent variation to the measurement.
Translocation control by hybridization (TCH) is used herein to refer to a method to pause a nanopore translocation event by using a structure created by hybridization which disassociates for translocation to proceed. Akeson et al. (U.S. Pat. No. 6,465,193) first demonstrated this by pausing DNA translocation with sequential hairpin duplexed regions. Translocation stopped at the duplex because it was larger than the α-hemolysin nanopore aperture. When the duplex released due to stochastic thermal fluctuation, translocation proceeded to the next duplex. During each pause, the region of DNA located in the nanopore (adjacent to the duplex) could be measured and identified. Akeson measured translocations of molecules with sequential duplexed regions and identified homogeneous regions adjacent to the duplexes. He estimated the mean of the stochastic pauses to be 15 to 18 μs per base pair for hairpin duplexes of 2 to 10 base pairs. Akeson further proposed that similar methods of pausing could be accomplished with alternative non-DNA structures.
Meller et al. (U.S. Pat. No. 7,972,858) used TCH to sequentially pause the translocation of the DNA with a series of duplexes formed by hybridizing complimentary oligomers to regions along the DNA. This technique utilized an optical technique to measure the each type of duplex that was sequentially released.
Gundlach et al. also used complementary oligomers for TCH (which he called “duplex interrupted”) to measure DNA designed with multiple duplex regions using a low noise nanopore derived from Mycobacterium smegmatis (“MspA”) (Proc. Natl. Acad. Sci., 2010). In this case the duplexes were formed by free strand hybridization and paused an adjacent 3-base homopolymer portion in the MspA constriction for measurement. Under such conditions three-base homopolymer identification was demonstrated. The complementary oligomers used for duplexing were 14 bases long, but had poor hybridization fill rates of only 65%. For sequencing, this approach requires DNA conversion methods such as those disclosed by Kokoris et al. or Meller et al. (supra).
In addition, groups have previously used duplexes to hold and release molecules form nanopores for a variety of applications, for example: (i) F. Sauer-Budge et. al., “Unzipping Kinetics of Double-Stranded DNA in a Nanopore,” Phys. Rev. Lett., vol. 90, no. 23, p. 238101, June 2003; (ii) N. Ashkenasy, J. Sánchez-Quesada, M. R. Ghadiri, and H. Bayley, “Recognizing a Single Base in an Individual DNA Strand: A Step Toward Nanopore DNA Sequencing,” Angew Chem Int Ed Engl, vol. 44, no. 9, pp. 1401-1404, February 2005; (iii) S. Howorka and H. Bayley, “Probing Distance and Electrical Potential within a Protein Pore with Tethered DNA,” Biophysical Journal, vol. 83, no. 6, pp. 3202-3210, December 2002; and (iv) W. A. Vercoutere, S. Winters-Hilt, V. S. DeGuzman, D. Deamer, S. E. Ridino, J. T. Rodgers, H. E. Olsen, A. Marziali, and M. Akeson, “Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules,” Nucleic Acids Res, vol. 31, no. 4, pp. 1311-8, February 2003.
While significant advances have been made in this field, commercially viable implementation of duplex translocation control with, for example, Xpandomers would benefit from improvements that overcome limitations caused by duplexing, including: (i) compositions for reporters to provide low noise, ion current blockage signals with amplitudes distributed for distinct high signal-to-noise measurements in nanopores; (ii) compositions that provide for uniform TCH release rates; (iii) improving poor hybridization fill rate on Xpandomer TCH duplex sites (ideally approaching 100% since each missed duplex can lead to lost sequence information); (iv) improving or mitigating lost dynamic range due to ion current blockage of the duplex at the nanopore entrance; and/or (v) methods to better utilize nanopore measurement bandwidth that is limited by the stochastics of duplex dissociation.
The present invention fulfills these needs and provides further related advantages as discussed below.